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CN-122029187-A - VHH-CH3 fusion proteins

CN122029187ACN 122029187 ACN122029187 ACN 122029187ACN-122029187-A

Abstract

The present invention provides improved constructs comprising immunoglobulin-domains. In particular, the invention provides novel VHH-CH 3 fusion antigen-binding forms, and improved methods of producing VHH-CH 3 fusions.

Inventors

  • J. P. Bogen
  • J. Wusterhube Lausch
  • H-u. Schmidt

Assignees

  • 百欧恩泰欧洲股份公司

Dates

Publication Date
20260512
Application Date
20240906
Priority Date
20230906

Claims (16)

  1. 1. A construct, comprising: a) A first polypeptide comprising one or more constant region immunoglobulin domains and one or more variable region immunoglobulin domains N-terminal to the constant region immunoglobulin domains, wherein all of the constant region immunoglobulin domains in the first polypeptide are heavy chain constant region 3 (CH 3) domains and all of the variable region immunoglobulin domains N-terminal to the CH3 domains are single domain heavy chain variable region immunoglobulin (VHH) domains.
  2. 2. The construct of claim 1, further comprising: b) A second polypeptide comprising one or more constant region immunoglobulin domains and one or more variable region immunoglobulin domains N-terminal to the constant region immunoglobulin domains, wherein all of the constant region immunoglobulin domains in the second polypeptide are CH3 domains and all of the variable region immunoglobulin domains N-terminal to the CH3 domains are VHH domains.
  3. 3. The construct of claim 1 or 2, wherein the first polypeptide comprises a single CH3 domain and the second polypeptide, if present, comprises a single CH3 domain.
  4. 4. The construct of any one of claims 1 to 3, wherein the first polypeptide comprises a single VHH domain N-terminal to the CH3 domain and the second polypeptide, if present, comprises a single VHH domain N-terminal to the CH3 domain.
  5. 5. The construct of any one of claims 2 to 4, wherein one or more of the CH3 domains of the first polypeptide and/or one or more of the CH3 domains of the second polypeptide comprises a modification that enhances formation of a construct comprising the first and second polypeptides.
  6. 6. The construct of any one of claims 2 to 5, wherein the CH3 domain of the first polypeptide forms a disulfide bond with the CH3 domain of the second polypeptide.
  7. 7. The construct of any one of claims 2 to 6, wherein: a) The sequence of the first polypeptide between the CH3 domain of the first polypeptide and the VHH domain located N-terminally of the CH3 domain in the first polypeptide consists of a first linking sequence, and B) The sequence of the second polypeptide between the CH3 domain of the second polypeptide and the VHH domain located N-terminal to the CH3 domain in the second polypeptide consists of a second linking sequence.
  8. 8. The construct of claim 7, wherein the first linking sequence and the second linking sequence do not form disulfide bonds.
  9. 9. The construct of claim 8, wherein one or more of the CH3 domains of the first polypeptide and/or one or more of the CH3 domains of the second polypeptide comprises a modification that enhances formation of a construct comprising the first polypeptide and second polypeptide, preferably wherein the CH3 domain of the first polypeptide forms a disulfide bond with the CH3 domain of the second polypeptide.
  10. 10. One or more nucleic acid sequences capable of expressing a construct according to any one of claims 1 to 9, a first polypeptide as defined in any one of claims 1 to 9 or a second polypeptide as defined in any one of claims 2 to 9.
  11. 11. A nucleic acid particle comprising the construct or nucleic acid sequence of any one of claims 1 to 10.
  12. 12. A cell comprising the construct, nucleic acid sequence or nucleic acid particle of any one of claims 1 to 11.
  13. 13. A composition comprising a construct, nucleic acid sequence, nucleic acid particle or cell according to any one of claims 1 to 12.
  14. 14. An in vitro method comprising contacting a cell with a construct, nucleic acid sequence, nucleic acid particle, cell or composition according to any one of claims 1 to 13.
  15. 15. Construct, nucleic acid sequence, nucleic acid particle, cell or composition according to any one of claims 1 to 13 for use in a therapeutic or diagnostic method.
  16. 16. A method of making a construct, wherein the method comprises: (i) Providing a first polypeptide comprising one or more constant region immunoglobulin domains and one or more variable region immunoglobulin domains N-terminal to the constant region immunoglobulin domains, wherein all of the constant region immunoglobulin domains in the polypeptide are CH3 domains, wherein all of the variable region immunoglobulin domains N-terminal to the CH3 domains are VHH domains, and wherein the sequence between the CH3 domains and the VHH domains located N-terminal to the CH3 domains consists of a first linking sequence; (ii) Providing a second polypeptide comprising one or more constant region immunoglobulin domains and one or more variable region immunoglobulin domains N-terminal to said constant region immunoglobulin domains, wherein all of said constant region immunoglobulin domains in said polypeptide are CH3 domains, wherein all of said variable region immunoglobulin domains N-terminal to said CH3 domains are VHH domains, and wherein the sequence between said CH3 domains and the VHH domain N-terminal to said CH3 domains consists of a second linking sequence, and (Iii) A construct comprising the first polypeptide and the second polypeptide is formed by forming a disulfide bond between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide without forming a disulfide bond between the first linker sequence and the second linker sequence.

Description

VHH-CH3 fusion proteins Technical Field The present invention provides novel forms of immunoglobulin domain-containing proteins, namely single domain heavy chain variable region (VHH) -heavy chain constant region 3 (CH 3) immunoglobulin domain fusions, comprising one or more VHH domains fused to the N-terminus of a CH3 immunoglobulin domain, either directly or via a linker sequence. The invention also relates to improved VHH-CH3 fusions and methods of making the same, which result in reduced aggregation and improved production of VHH-CH3 fusions. Background Monoclonal antibodies (mabs) are the fastest growing class of biological agents, with more than 100 molecules approved for therapeutic use. Despite its generally advantageous biophysical properties, high specificity, long half-life, and low immunogenicity, full-length mabs suffer from several disadvantages. Including limited tumor accumulation and penetration due to its size (Yokota et al, cancer Res.,1992,52:3402-8) and single epitope selectivity. Bispecific antibodies (bsAb) have attracted considerable interest in the scientific community over the last decade, where many different formats have been tested for a variety of applications. In typical antibodies, the binding portion consists of VH and VL domains, whereas camelid antibodies exhibit a unique antibody subtype comprising only heavy chains, known as heavy chain-only antibodies (HcAb). The binding portion in these camelid antibodies consists of a single domain, which is referred to as the heavy chain variable region domain (VHH, also referred to herein as "single domain heavy chain variable region immunoglobulin") domain of the heavy chain. This single domain antibody (sdAb) is about the size of a human VH (15-kDa) and is of smaller size than Fab fragments or scFv because it does not require VL domains for antigen recognition and stable expression. This smaller size allows deeper tumor penetration (Sun et al Int J Nanomedicine,2021,16:2337-2356; jovEvska et al BioDrugs,2020,34:11-26; chakravarty et al Theranostics,2014,4:386-98; and Kijanka et al Nanomedicine (Lond), 2015,10:161-74). Some studies even report VHH crossing the blood brain barrier. However, similar to fragments of conventional antibodies, VHHs, which are separate binding entities, are limited by their short half-lives. US2023/242676A1 relates to specific antibody dimerization domains and discloses that the dimerization domain of the patent application may comprise a CH2 domain, a CH3 domain or a combination thereof. The antibody construct of this patent application comprises a full length Fc region (i.e., comprising both CH2 and CH3 domains), as illustrated by the figures. WO2022/143801A1 discloses antibody constructs specifically described as comprising CH3 and CH2 domains or full length Fc regions. WO2022/150785A2 discloses antibody constructs comprising modified CH3 domains. Constructs are not illustrated as comprising a CH3 domain as the only constant domain of an antibody. Figures 3 and 8 of WO2022/150785A2 contain a speculative illustration of constructs comprising only CH3 constant domains, but these illustrated constructs comprise both VL and VH domains and thus do not comprise VHH domains. WO2008/071685A1 relates to nanobodies. WO2008/071685A1 is a speculative disclosure, working example free. As published papers, nanobodies are pointed out that can be "linked" to CH1, CH2 and/or CH3 domains, but the nature and direction of the linkage (linkage) is not mentioned. Conventional full-length mabs comprise a fragment crystallizable (Fc) region that is a homodimer of the CH2-CH3 domain. Although the CH2 domain is bound by fcγ receptor (fcγr) and mediates effector functions of antibodies, recognition of the elbow region between CH2 and CH3 by neonatal Fc receptor (FcRn) is responsible for the long plasma half-life of IgG1 antibodies. Great efforts have been made to engineer the Fc region to extend or shorten the half-life of antibodies or to increase or silence effector functions. However, in terms of molecular weight, the Fc region still occupies a large portion of the entire molecule. This in turn limits tumor penetration of the antibody. Various antibody formats (e.g., scFv, diabodies, fab, F (ab') 2, and others) lacking an Fc region have been developed to produce smaller binding molecules, attempting better tumor penetration. However, its small size also tends to result in rapid renal clearance. Thus, while these molecules may in principle allow deeper tumor penetration, the limited plasma half-life prevents strong enrichment in the tumor and reduces its effectiveness. In an attempt to produce molecules with moderate half-life and allowing strong tumor accumulation, minibodies and Δch2 IgG were introduced in the 1990 s. Minibodies consist of scFv linked to part of the hinge region of IgG1 antibodies and to the CH3 domain of IgG1 antibodies via glycine-serine-linkers. Δch2 IgG exhibits a similar architecture using Fab instead o